This invention relates to a method for immunoassay and reagents therefor, particularly to an analytical method and reagents therefor, which are suitable for assaying an antigen or an antibody in a body fluid sample by a polarization fluorescence method.
The previously known methods for measuring the concentration of an antigen or an antibody in a body fluid sample by fluorometry concern a method using microcapsules and a method utilizing polarization fluorescence. The method using microcapsules is shown, for example, in Japanese Patent Application Kokai (Laid-Open) No. 117159/85. In this method, carboxyfluorescein is encapsulated as a fluorescent compound in liposomes labeled with an antigen an antibody. This fluorescent compound undergoes self-quenching when its concentration is high, therefore by utilizing this property, it can be kept quenched while encapsulated in the liposomes. When the liposomes are lysed due to complement activated by the immune complexes resulting from the antigen-antibody reactions, the fluorescent compound flows out into the liquid outside the liposomes thereby decreasing in concentration and emitting fluorescence. The concentration of an antigen or an antibody in a sample is determined by measuring the intensity of the fluorescence produced.
Such a conventional method using microcapsules is not widely applicable because only fluorescent substances having self-quenching properties can be used.
On the other hand, an example of the method utilizing polarization fluorescence is shown, for example, in U.S. Pat. No. 4,429,230.
When fluorescent molecules are excited by polarized light, the orientation of the excited molecules is abolished by rotation of the molecules by Brownian motion during the period between light absorption and emission, so that the degree of polarization of fluorescence is lowered. When fluorescence intensities obtained when an polarizing plate on the exciting light side is vertically fixed and a polarizing plate on the fluorescence side is placed parallel or perpendicular thereto are taken as I.sub..parallel. and I.sub..perp. respectively, the degree of polarization fluorescence P is expressed by the formula: ##EQU1##
When an antigen labeled with a fluorescent substance binds to the corresponding antibody, the molecules greatly increase in size, resulting in depression of rotary Brownian motion of the fluorescent molecules, and hence the degree of polarization becomes larger than that before the binding to the antibody. An assay utilizing the above-mentioned principle is polarization fluoroimmunoassay, which is as follows. When a fluorescence-labeled antigen is first reacted with an antibody and an unlabeled antigen (an unknown substance) to be determined is added to this system, the antibody is consumed so that the amount of labeled antigen binding to the antibody is reduced, and the amount of labeled antigen which does not bind to the antibody is increased. Since this labeled antigen has a low molecular weight, its rotary Brownian motion occurs actively, so that the degree of polarization is reduced. Therefore, the amount of the unknown substance can be determined from the extent of reduction of the degree of polarization.
In the case of the conventional immunoassay utilizing polarization fluorescence which is shown above, and in U.S. Pat. No. 4,429,230, the intensity of emission of fluorescence is low. Hence, it has been impossible to attain a high S/N ratio, wherein S indicates Signal and N indicates Noise.